The bone. The solid matrix of the bone is made of an organic phase, collagen, and of an inorganic phase, composed of calcium and phosphate (hydroxyapatite). Bones are continuously remodeling themselves, a process of combined dissolution (resorbtion) and re-construction of the bone matrix. The bone relies on two cell types for remodeling process: osteoclasts, which are responsible for resorbtion, and osteoblasts, which promote for bone formation. In normal bones, both processes are strictly coordinated to maintain bone mass within defined limits. More specifically, at a specific site, resorbtion occurs first and over a shorter period than bone formation. At the end of the formation stage most osteoblasts will disappear through the apoptotic process. About 10% will be enclosed in the bone and remain there to form osteocytes. It is generally believed that the osteocytes participate in some form of mechanical stress sensing mechanism. Osteoporosis occurs when the resorbtion process is initiated more often than formation is completed, leading progressively to more fragile bones (Harisson 14th Ed). PHEX. The PHEX gene (formerly PEX; a Phosphate regulating gene with homologies to Endopeptidases on the X chromosome) was identified by a positional cloning approach as the candidate gene for human X-linked hypophosphatemia (XLH) (1). XLH is a Mendelian disorder of phosphate homeostasis characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal defects in phosphate re-absorption and vitamin D metabolism (2). Several groups have cloned and sequenced the human and mouse PHEX/Phex cDNAs (3-7) (PHEX/Phex refers to the human and mouse genes, respectively). Amino acid sequence comparisons have demonstrated homologies between PHEX/Phex protein and members of the M13 endopeptidase family, as previously observed in the partial sequence of the candidate gene (1). The M13 endopeptidases are zinc-containing type 11 integral membrane glycoproteins with a relatively short cytoplasmic amino-terminal region, a single transmembrane domain, and a long extracytoplasmic domain, which contains the active site of the enzyme (8). In addition to PHEX, this family includes neprilysin (NEP, neutral endopeptidase 24.11), a widely distributed peptidase involved in the degradation of several bioactive peptides (9), the endothelin-converting enzymes 1 and 2 (ECE-1 and ECE-2) responsible for the processing of inactive big-endothelins into active endothelins (10), the Kell blood group protein, a protein of the erythrocyte membrane with unknown function (11), and ECEL/DINE (12,13) and SEP/NL1 (14,15), two recently reported peptidases with homology to the neprilysin family.
The precise physiological role of PHEX is unknown and the mechanisms whereby loss of PHEX function causes renal phosphate wasting, abnormal regulation of vitamin D metabolism and impaired bone mineralization are not completely understood. Homology of PHEX to members of the M13 family of zinc metallopeptidases suggests a role in regulating the activity of extra-cellular bioactive peptide(s) that act in an autocrine, paracrine or endocrine fashion. In support of this hypothesis, Lajeunesse et al. (16) and Nesbitt et al. (17) have reported the existence of a renal phosphate transport inhibitory factor in the culture medium of osteoblasts isolated from the Hyp mouse, an animal model for human XLH (18). The role of PHEX would appear be to inactivate this circulating factor. However, the molecular identity of this factor (phosphatonin) has yet to be established.
PHEX and mineralization. In situ hybridization performed on sections of embryos and newborn mice showed the presence of Phex mRNA in osteoblasts and odontoblasts (32). Phex gene expression was detectable on day 15 of embryonic development, which coincides with the beginning of intracellular matrix deposition in bones. Moreover, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the Phex transcript is decreased in adult bones and in non growing teeth but maintained in growing incisors throughout life. This result was confirmed when the presence of the Phex protein in new born and adult bones was investigated by Western blotting using a monoclonal antibody raised against human PHEX. Immunohistochemical studies on a 2 month-old mouse showed exclusive labeling of mature osteoblasts and osteocytes in bones and of odontoblasts in teeth (27). Taken together these results suggest that PEX/Phex plays an important role in the development and maintenance of mineralization in these tissues.
Studies performed with the Hyp mouse, an animal model harboring a large deletion in the 3′ region of the Phex gene (5) and exhibiting the same phenotypic features that characterize patients with XLH (18), also suggest that PHEX is involved, in an unknown way, in the mineralization process. Hyp mice exhibit enlarged osteoid area in bones (27, 33) which was shown not to be due to abnormal matrix deposition (34,35) but to impaired mineralization (33).
Osteocalcin. Osteocalcin is the most abundant of the non-collagenous bone proteins and is expressed only in osteoblasts. The mature peptide is 49 amino acids long with 3 Gla residues and one difulfide bond. A vitamin K-dependent gamma-carboxylase is responsible for the transformation of certain Glu into Gla residues. These Gla residues are well known for their affinity toward calcium ions and hydroxyapatite crystals. About 80% of bone mass is made-up of hydroxyapatite, a mineral composed of calcium and phosphate. An increase in osteocalcin mRNA is associated with the mineralization stage and the transformation of osteoblasts into osteocytes (39).
In vitro, osteocalcin inhibits growth of mineral crystals (36, 37) whereas in vivo, gene targeting aimed at the disruption of both mouse osteocalcin genes resulted in the generation of osteocalcin-deficient animals (38). These osteocalcin-knockout mutant mice display a phenotype “opposed to osteoporosis” which is also characterized by an increase in the rate of bone formation, increased bone mass, as well as an overall improved functional quality of the bone.